The mitochondrial F1F0 ATP synthase catalyzes synthesis of the vast majority of ATP that is utilized by mammalian cells, in the culmination of an intricate process known as oxidative phosphorylation. It is a multi- subunit, membrane-bound enzyme that is known to function with rotary motion of some of its subunits. Mutations found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzyme are found in chloroplasts and in the plasma membranes of some bacteria. Many of the recent insights into the structure and function of the ATP synthase have come from studies of the E. coli enzyme. This version of the enzyme contains eight different types of subunits. Mutations found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzyme are found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzymes are found in chloroplasts and in the plasma membranes of some bacteria. Many of the recent insight into the structure and function of the ATP synthase have come from studies of the E. coli enzyme. This version of the enzyme contains eight different types of subunits. Alpha, beta, gamma, delta, and epsilon form F1, containing the sites of ATP synthesis. Subunits a, b and c form the membrane sector F0, containing the proton pathway. The movement of protons through F0 is thought to drive the rotation of gamma and epsilon subunits, relative to the alpha and beta subunits, which form the ATP catalytic sites. The studies proposed in this application focus on two of the subunits from the E. coli ATP synthase, epsilon and subunit a. Four specific aims will be pursued. (A) Putative functional regions of subunit a will be examined. Transmembrane spans will be probed by alanine insertion scanning mutagenesis and conserved residues will be mutated for examining of effects on function. (B) Important structural features of subunit a will be identified. Near- neighbor relationships of transmembrane spans in subunit a will be established, and the putative "half-channels" will be tested by labeling procedures. (C) Subunit interactions among F0 subunits will be investigated by photoactive crosslinking from Cys residues. (D) Structural issues in the epsilon subunit that relate to function will be examined. This includes the surface of epsilon involved in binding to other subunits in the ATP synthase, and the role of flexibility within the epsilon subunit for function.